This proposal has two long-term goals. The first is an understanding, in precise molecular terms, of the mechanism of action of highly conserved repressor of gene transcription. This work will be carried out with the S. cerevisiae Ssn6-Tupl repressor and emphasizes the use of purified proteins for biochemical and structural approaches designed to deduce molecular mechanisms. Genetic approaches in S. cerevisiae, which include the extensive use of microarray technologies will be used to test models derived from biochemical experiments and as exploratory tools. The second long-term goal is a description of the entire Ssn6-Tupl regulatory network in S. cerevisiae and several related yeasts, including all the DNA-binding proteins that utilize Ssn6-Tupl and the complete sets of genes controlled by each DNA-binding protein. The ultimate goal of this work is an understanding of how regulatory circuits that are composed of highly conserved components become specialized over evolutionary time scales. Given the high degree of conservation of gene regulatory proteins and general transcription factors among eukaryotes, it seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply in other settings. A basic understanding of the molecular events underlying cell specialization provides not only a framework for understanding how the process can fail, but also provides the substrates and knowledge to design therapeutic strategies based on intervention.